The Syk-binding ubiquitin ligase c-Cbl mediates signaling-dependent B cell receptor ubiquitination and B cell receptor-mediated antigen processing and presentation

Abstract

B cell receptor (BCR)-mediated antigen (Ag) processing and presentation lead to B cell-T cell interactions, which support affinity maturation and immunoglobulin class switching. These interactions are supported by generation of peptide-MHC class II complexes in multivesicular body-like MIIC compartments of B cells. Previous studies have shown that trafficking of Ag·BCR complexes to MVB-like MIIC occurs via an ubiquitin-dependent pathway and that ubiquitination of Ag·BCR complexes occurs by an Src family kinase signaling-dependent mechanism that is restricted to lipid raft-resident Ag·BCR complexes. This study establishes that downstream Syk-dependent BCR signaling is also required for BCR ubiquitination and BCR-mediated antigen processing and presentation. Knockdown studies reveal that of the two known Syk-binding E3 ubiquitin ligases c-Cbl and Cbl-b, only c-Cbl appears to have a central role in BCR ubiquitination, trafficking to MIIC, and ubiquitin-dependent BCR-mediated antigen processing and presentation. These results establish the novel role for Syk signaling and the Syk-binding ubiquitin ligase c-Cbl in the BCR-mediated processing and presentation of cognate antigen and define one mechanism by which antigen-induced BCR ubiquitination is modulated to impact the initiation and maturation of the humoral immune response.

FIGURE 1.

Inhibition of Syk activity abrogates BCR ubiquitination. A, fluo-3- and Fura Red-loaded A20μWT B cells were pretreated with the indicated dose of piceatannol or diluted vehicle (DMSO), stimulated with anti-human IgM antibody, and then the resulting intracellular calcium flux was determined by flow cytometry. Shown are representative traces from one of three independent experiments. B, graphical representation of the ubiquitin (Ubi) pull-down approach. C, A20μWT B cells were pretreated for 1 h at 37 °C with indicated concentrations of piceatannol (Pice) or diluted vehicle (DMSO) and then pulsed with anti-human IgM-btn (anti-BCR) for the 20 min. Cells were then lysed, and a fraction of whole cell lysate was retained. Ubiquitinated ligand-BCR complexes were isolated from the remaining whole cell lysate by UQ1 pull-down. BCR (Total BCR from the whole cell lysate and ubiquitinated BCR and Ubi-BCR from the UQ1 pull-down) was detected by SDS-PAGE and anti-IgM Western blot analysis. Ligand-BCR (Total Ag·BCR from the whole cell lysate and ubiquitinated Ag·BCR and Ubi-Ag·BCR from the UQ1 pull-down) was detected by SDS-PAGE and SA-HRP Western blot analysis. GAPDH serves as a loading control for total BCR/Ag·BCR. Shown are representative results from one of three independent experiments. D, A20μWT B cells were pretreated for 1 h at 37 °C with 10 μm piceatannol or diluted vehicle (DMSO) and then pulsed with anti-human IgM-btn for the indicated times. Cells were then lysed, and total Ag·BCR and ubiquitinated ligand-BCR complexes were detected by SDS-PAGE and blotting with SA-HRP as in C. GAPDH was used as a loading control. Shown are representative results from one of three independent experiments.

FIGURE 2.

BCR-mediated antigen presentation requires Src-family kinase and Syk signaling. A, splenocytes from MD4.B10.Br (black bars, BCR-mediated) or B10.Br (white bars, fluid-phase mediated) mice were preincubated for 1 h with indicated inhibitors or diluted vehicle (DMSO). Cells were then pulsed with antigen (MD4.B10.Br, 100 nm HEL for BCR-mediated processing; B10.Br, 100 μm HEL + 10 μg/ml anti-murine IgM for F-P processing and BCR signaling) for 24 h, harvested, and HEL_46–61-I-A^k complex expression was determined by staining with the HEL_46–61-I-A^k complex-specific mAb C4H3 and analysis flow cytometry. Shown is the normalized MFI of C4H3 staining of B220+ cells, mean ± 1 S.E., for three independent experiments. PP1, 4-amino-5-(4-methylphenyl)-7-(t-butyl) pyrazolo [3,4-d]-pyrimidine; PP2, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine. B, parallel samples were stained with a pan-reactive anti-I-A^k monoclonal antibody (10–3.6-FITC) and analyzed by flow cytometry. Shown is the normalized MFI of 10–3.6 staining of B220+ cells, mean ± 1 S.E. for three independent experiments. Each experimental sample was compared with the vehicle (DMSO) control by a Student's t test. *, p ≤ 0.05. Pice, piceatannol.

FIGURE 3.

Stable knockdown of the ubiquitin E3 ligases c-Cbl and Cbl-b in A20μWT B cells. A, domain organization and comparison of c-Cbl and Cbl-b E3 ubiquitin ligases. There is 50% similarity between the two proteins at the amino acid level and 55% similarity at the nucleotide level, with most of the differences between the two proteins restricted to the C-terminal ends of the proteins. The tyrosine kinase binding (TKB) domain is responsible for binding the Tyr-323 phosphorylated form of Syk. #1, #2, #3 indicate the shRNA sense binding sites. B, SDS-PAGE and anti-Cbl Western blot analysis of whole cell lysates. Densitometry results (derived from Western blot analyses from three independent experiments) were normalized first to the loading control β-actin and then to the level of Cbl in WT control cells. Student's t test was used to compare the level of Cbl in knockdown versus WT cells. *, p ≤ 0.05.

FIGURE 4.

The effect of selective Cbl knockdown on BCR signaling and endocytosis. A and B, fluo-3- and Fura Red-loaded B cells were stimulated with anti-human IgM antibody (A) or calcium ionophore (B), and the resulting intracellular calcium flux was monitored by flow cytometry. BCR-elicited calcium signaling is selectively increased in Δc-Cbl B cells. Shown are representative tracings from one of three independent experiments. C, biotinylated anti-huIgM was bound to B cells on ice. The cells were washed, incubated at 37 °C for the indicated time, and then the fraction of anti-BCR-btn left at the surface was determined by SA-Alexa Fluor 488 staining and analysis by flow cytometry. Shown is the average level of surface ligand at each time point from three independent experiments (p = 0.75).

FIGURE 5.

c-Cbl mediates Ag·BCR ubiquitination and processing. Upper panel, B cells were pulsed with anti-human IgM-btn for the indicated time (minutes) at 37 °C. The cells were lysed and ubiquitinated ligand-BCR complexes isolated by ubiquitin pull-down of a fraction of the whole cell lysate (Fig. 1C). The level of total ligand-BCR complexes (Ag·BCR) and ubiquitinated ligand-BCR complexes (Ag·BCR-Ubi) was determined by SDS-PAGE and SA-HRP blotting of whole cell lysate and ubiquitin pull-down, respectively. The numbers below the blots represent the normalized level of total IgM heavy chain detected in each sample (average from three independent experiments). Lower panel, longer exposure of SA-HRP blots of whole cell lysate (also probed with anti-β-actin antibody). HC and LC, intact heavy and light chains of anti-BCR-btn antibody; Deg bands, degradation bands of anti-BCR-btn HC).

FIGURE 6.

c-Cbl mediates Ag·BCR trafficking to LAMP-2⁺ endocytic compartments. A, B cells were pulsed on ice with 10 μg/ml anti-huIgM-btn followed by SA-Alexa Fluor 594, washed, and then incubated at 37 °C for 30 min. Cells were then fixed, permeabilized, and stained with anti-CD107b (LAMP-2). BCR staining is shown in red, LAMP-2 in pseudo-color green, and DAPI in blue. Shown are representative results from one of three independent experiments. Scale bar = 10 μm. B, for each cell type, the Pearson's coefficient of BCR-LAMP-2 colocalization at 30 min was measured for 100–150 cells. Shown is the average Pearson's coefficient of BCR-LAMP-2 colocalization for three independent experiments (mean ± 1 S.E.). Student's t test was used to compare the Pearson's coefficient of knockdown versus WT cells. *, p ≤ 0.05.

FIGURE 7.

c-Cbl is essential for BCR-mediated antigen presentation. The indicated B cells (all expressing a PC-specific BCR) were cocultured with DO-11.10 T cells and the indicated dose of either PC-Ova (for BCR-mediated antigen processing) or Ova (for fluid-phase antigen processing). An ELISA was used to determine the level of DO-11.10 produced IL-2 in the culture supernatants. Shown are representative results from one of three independent experiments. Values are an average of three measurements. Presentation of PC-Ova is significantly different (p < 0.01) from that of Ova in all cells except Δc-Cbl B cells.